A Mutation in the SH2 domain of STAT2 prolongs tyrosine phosphorylation of STAT1 and promotes type I IFN-induced apoptosis

Type I interferons (IFN-alpha/beta) induce apoptosis in certain tumor cell lines but not others. Here we describe a mutation in STAT2 that confers an apoptotic effect in tumor cells in response to type I IFNs. This mutation was introduced in a conserved motif, PYTK, located in the STAT SH2 domain, which is shared by STAT1, STAT2, and STAT3. To test whether the tyrosine in this motif might be phosphorylated and affect signaling, Y631 of STAT2 was mutated to phenylalanine (Y631F). Although it was determined that Y631 was not phosphorylated, the Y631F mutation conferred sustained signaling and induction of IFN-stimulated genes. This prolonged IFN response was associated with sustained tyrosine phosphorylation of STAT1 and STAT2 and their mutual association as heterodimers, which resulted from resistance to dephosphorylation by the nuclear tyrosine phosphatase TcPTP. Finally, cells bearing the Y631F mutation in STAT2 underwent apoptosis after IFN-alpha stimulation compared with wild-type STAT2. Therefore, this mutation reveals that a prolonged response to IFN-alpha could account for one difference between tumor cell lines that undergo IFN-alpha-induced apoptosis compared with those that display an antiproliferative response but do not die.     

Advancing salt marsh restoration for coastal resilience: a learning exchange

A multidisciplinary group of salt marsh professionals from Maine to Virginia participated in a collaborative learning exchange to improve restoration for the overall health and resilience of coastal wetlands. This was an unprecedented forum through which participants representing different geographies, backgrounds, and roles in salt marsh management were able to share and learn from one another to develop the best available restoration methods for on-the-ground projects that address multiple benefits. By including mosquito control agencies, restoration practitioners, regulatory agencies, academic researchers, and conservation organizations in the learning exchange, we developed an understanding and acceptance of different approaches. Regulators learned about project ideas and contributed to project designs in early development stages. Collaborating while engaged in on-the ground projects enabled participants to implement lessons learned in real time. Field trips to restoration sites at different stages of development allowed a greater and more fluid exchange of ideas and practical implementation advice. Practitioners leveraged resources and developed new collaborations. Lessons learned and shared through this faster and more flexible forum will inform the design, implementation, and monitoring of restoration projects across the region and improve overall marsh health and resilience in the face of climate change. Learning exchanges like this should be used more frequently to improve the efficiency and effectiveness of coastal restoration particularly when there is a windfall of cash and a short window of opportunity such as with post-disaster federal spending.

     

Modulation of inducible nitric oxide synthase (iNOS) expression and cardiovascular responses during static exercise following iNOS antagonism within the ventrolateral medulla

Previous reports indicate that inducible nitric oxide synthase (iNOS) blockade within the rostral ventrolateral medulla (RVLM) and caudal ventrolateral medulla (CVLM) differentially modulated cardiovascular responses, medullary glutamate, and GABA concentrations during static skeletal muscle contraction. In the current study, we determined the role of iNOS antagonism within the RVLM and CVLM on cardiovascular responses and iNOS protein expression during the exercise pressor reflex in anesthetized rats. Following 120 min of bilateral microdialysis of a selective iNOS antagonist, aminoguanidine (AGN; 10 µM), into the RVLM, the pressor responses were attenuated by 72 % and changes in heart rate were reduced by 38 % during a static muscle contraction. Furthermore, western blot analysis of iNOS protein abundance within the RVLM revealed a significant attenuation when compared to control animals. In contrast, bilateral administration of AGN (10 µM) into the CVLM augmented the increases in mean arterial pressure by 60 % and potentiated changes in heart rate by 61 % during muscle contractions, but did not alter expression of the iNOS protein within the CVLM. These results demonstrate that iNOS protein expression within the ventrolateral medulla is differentially regulated by iNOS blockade that may, in part, contribute to the modulation of cardiovascular responses during static exercise.     

Effectiveness of interventions designed to reduce the use of imaging for low-back pain: a systematic review

Background:Rates of imaging for low-back pain are high and are associated with increased health care costs and radiation exposure as well as potentially poorer patient outcomes. We conducted a systematic review to investigate the effectiveness of interventions aimed at reducing the use of imaging for low-back pain.Methods:We searched MEDLINE, Embase, CINAHL and the Cochrane Central Register of Controlled Trials from the earliest records to June 23, 2014. We included randomized controlled trials, controlled clinical trials and interrupted time series studies that assessed interventions designed to reduce the use of imaging in any clinical setting, including primary, emergency and specialist care. Two independent reviewers extracted data and assessed risk of bias. We used raw data on imaging rates to calculate summary statistics. Study heterogeneity prevented meta-analysis.Results:A total of 8500 records were identified through the literature search. Of the 54 potentially eligible studies reviewed in full, 7 were included in our review. Clinical decision support involving a modified referral form in a hospital setting reduced imaging by 36.8% (95% confidence interval [CI] 33.2% to 40.5%). Targeted reminders to primary care physicians of appropriate indications for imaging reduced referrals for imaging by 22.5% (95% CI 8.4% to 36.8%). Interventions that used practitioner audits and feedback, practitioner education or guideline dissemination did not significantly reduce imaging rates. Lack of power within some of the included studies resulted in lack of statistical significance despite potentially clinically important effects.Interpretation:Clinical decision support in a hospital setting and targeted reminders to primary care doctors were effective interventions in reducing the use of imaging for low-back pain. These are potentially low-cost interventions that would substantially decrease medical expenditures associated with the management of low-back pain.Current evidence-based clinical practice guidelines recommend against the routine use of imaging in patients presenting with low-back pain.^1–3 Despite this, imaging rates remain high,^4,5 which indicates poor concordance with these guidelines.^6,7Unnecessary imaging for low-back pain has been associated with poorer patient outcomes, increased radiation exposure and higher health care costs.⁸ No short- or long-term clinical benefits have been shown with routine imaging of the low back, and the diagnostic value of incidental imaging findings remains uncertain.^9–12 A 2008 systematic review found that imaging accounted for 7% of direct costs associated with low-back pain, which in 1998 translated to more than US$6 billion in the United States and £114 million in the United Kingdom.¹³ Current costs are likely to be substantially higher, with an estimated 65% increase in spine-related expenditures between 1997 and 2005.¹⁴Various interventions have been tried for reducing imaging rates among people with low-back pain. These include strategies targeted at the practitioner such as guideline dissemination,^15–17 education workshops,^18,19 audit and feedback of imaging use,^7,20,21 ongoing reminders⁷ and clinical decision support.^22–24 It is unclear which, if any, of these strategies are effective.²⁵ We conducted a systematic review to investigate the effectiveness of interventions designed to reduce imaging rates for the management of low-back pain.     

Impact of IgG2 high molecular weight species on neonatal Fc receptor binding assays

A cell-based assay and a solution neonatal Fc receptor (FcRn) binding assay were implemented for the characterization of an IgG₂ antibody after observation that different product lots exhibited unexpected differences in FcRn binding in the cell-based format with membrane-bound FcRn. The experiments described here suggest that the apparent differences observed in the FcRn binding across different product lots in the cell-based format can be attributed to the different levels of the higher order high molecular weight species (HMWs) in them. A strong correlation between FcRn binding in the cell-based format and the percentage (%) higher order HMWs suggests that small amounts (∼0.1%) of the latter could cause the enhanced apparent FcRn binding (% relative binding ranging from 50 to 100%) in the format. However, when the binding was assessed with recombinant FcRn in soluble form, avidity effects were minimal and the assay format exhibited less sensitivity toward the differences in higher order HMWs levels across product lots. In conclusion, a solution-based assay may be a more appropriate assay to assess FcRn binding of the dominant species of an Fc-fusion protein or monoclonal antibody if minor differences in product variants such as higher order HMWs are shown to affect the binding significantly.     

Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors

Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.     

KUD773, a phenylthiazole derivative,displays anticancer activity in human hormone-refractory prostate cancers through inhibition of tubulin polymerization and anti-Aurora A activity

Background

Hormone-refractory prostate cancer (HRPC), which is resistant to hormone therapy, is a major obstacle in clinical treatment. An approach to inhibit HRPC growth and ultimately to kill cancers is highly demanded.

Results

KUD773 induced the anti-proliferative effect and subsequent apoptosis in PC-3 and DU-145 (two HRPC cell lines); whereas, it showed less active in normal prostate cells. Further examination showed that KUD773 inhibited tubulin polymerization and induced an increase of mitotic phosphoproteins and polo-like kinase 1 (PLK1) phosphorylation, indicating a mitotic arrest of the cell cycle through an anti-tubulin action. The kinase assay demonstrated that KUD773 inhibited Aurora A activity. KUD773 induced an increase of Cdk1 phosphorylation at Thr¹⁶¹ (a stimulatory phosphorylation site) and a decrease of phosphorylation at Tyr¹⁵ (an inhibitory phosphorylation site), suggesting the activation of Cdk1. The data were substantiated by an up-regulation of cyclin B1 (a Cdk1 partner). Furthermore, KUD773 induced the phosphorylation and subsequent down-regulation of Bcl-2 and activation of caspase cascades.

Conclusions

The data suggest that KUD773 induces apoptotic signaling in a sequential manner. It inhibits tubulin polymerization associated with an anti-Aurora A activity, leading to Cdk1 activation and mitotic arrest of the cell cycle that in turn induces Bcl-2 degradation and a subsequent caspase activation in HRPCs.     

Discovery of a novel series of selective HCN1 blockers

The discovery of a series of novel, potent, and selective blockers of the cyclic nucleotide-modulated channel HCN1 is disclosed. Here we report an SAR study around a series of selective blockers of the HCN1 channel. Utilization of a high-throughput VIPR assay led to the identification of a novel series of 2,2-disubstituted indane derivatives, which had moderate selectivity and potency at HCN1. Optimization of this hit led to the identification of the potent, 1,1-disubstituted cyclohexane HCN1 blocker, 2-ethoxy-N-((1-(4-isopropylpiperazin-1-yl)cyclohexyl)methyl)benzamide. The work leading to the discovery of this compound is described herein.